Abnormal expression of receptors in the Epidermal Growth Factor Receptor family, (the EGFR-family; also called the ErbB receptor family), is frequently associated with various malignancies in lung, breast, prostate, colon, ovary, head and neck. It is of interest to study this receptor family to gain a better understanding of the relation of the receptors to patient prognosis and treatment. The family consists of four transmembrane receptors, the epidermal growth factor receptor, EGFR, (ErbB1/HER1), HER2 (ErbB2/neu), HER3 (ErbB3) and HER4 (ErbB4) (Gullick W J. Endocr Rel Canc 2001; 8:75-82; Witton C. J. et al J Pathol 2003; 200:290-297). Each receptor comprises an extra-cellular ligand binding domain, a transmembrane domain and an intracellular tyrosine kinase domain (except HER3 which lacks a functional tyrosine kinase domain) (Citri A, et al. Exp Cell Res 2003; 284(1):54-65; Harari D and Yarden Y. Oncogene 2002; 19:6102-6114). There is one EGFR variant which has almost no ECD-EGFRvIII, Wikstrand C J et al Cancer Res. 55: 3140-3148, 1995; Huang H S et al J. Biol. Chem. 272: 2927-2935, 1997; Kuan C T, et al Endocr. Relat. Cancer 8: 83-96, 2001.
When a ligand binds to a receptor in the EGFR family, the receptor is stimulated to dimerise, either with another identical receptor (homodimerization) or with another receptor in the family (heterodimerization) (Olayioye M A, et al. Embo J. 2000; 19:3159-67; Yarden Y, Sliwkowski M X. Cell Biol 2001; 2:127-37). Receptor dimerization activates the intracellular tyrosine kinase domain, leading to proliferation, migration, apoptosis, differentiation or other cellular processes (Yarden Y, Sliwkowski M X. Cell Biol 2001; 2:127-37; Wells A. Int J Biochem Cell Biol 1999; 31:637-643; Vermeer P D et al. Nature 2003; 422:322-6). EGFR and HER2 are the most studied receptors of the four in the family and are over-expressed in many malignancies (Nordberg E et al. Eur J Nucl Med Mol. Imaging. 2005 July; 32(7):771-7). A high expression of these particular receptors is often associated with a poor prognosis (Hendriks B S et al. J Biol Chem 2003; 278:23343-23351; Arteaga C L. Oncologist 2002; 7 Suppl 4:31-9; Earp H S et al. Breast Cancer Res Treat 1995; 35:115-32; Wester K, et al. Acta Oncol 2002; 41:282-8. Lorenzo G D et al. Clin Prostate Cancer 2003; 2(1):50-7).
Several ligands bind to members of the EGFR receptor family. The only receptor that does not have any known natural ligand is HER2. (Citri A, et al. Exp Cell Res 2003; 284(1):54-65; Yarden Y, Sliwkowski M X. Cell Biol 2001; 2:127-37; Lenferink A E G, et al. EMBO J. 1998; 17:3385-3397). The antibody trastuzumab (Herceptin), which binds to the extra-cellular domain, may be used to target the HER2 receptor, especially in HER2 expressed tumors in breast cancer. Binding of trastuzumab can block growth stimulating intracellular signalling, decrease the capacity of cellular repair after chemo- and radiotherapy and possibly also improve the capacity of apoptosis. Bookman M A et al. J Clin Oncol 2003; 21:283-290; Pegram M D et al. Cancer Treat Res 2000; 103:747-75; McKeage K, Perry C M. Drugs 2002; 62:209-43). Affibody molecules disclosed in WO2005/003156 may also be used to target HER2.
EGFR function can be inhibited by blocking ligand binding to the extra-cellular part of the receptor, using antibodies such as cetuximab (Erbitux, ImClone/Bristol Myers Squibb) (Baselga J. Eur J Cancer 37: Suppl 4, S16-22, 2001, ABX-EGF Ranson M, Curr Opin Mol Ther 5: 541-546, 2003 or mab425/EMD55900 (Merck) or antibody fragments (Boskovitz A et al: Expert Opin Biol Ther 4: 1453-1471, 2004). The receptor function may in some, but not all patients, also be blocked with low molecular weight tyrosine kinase inhibitors such as Iressa (Gefitinib, AstraZeneca) (Sundberg A L et al: Eur J Nucl Med Mol Imaging 30: 1348-1356, 2003; Herbst R S et al: Nat Rev Cancer 4: 956-965, 2004) or Tarceva (Erlotinib, OSI-774) (Krozely P. Clin J Oncol Nurs 8: 163-168, 2004) that bind the intracellular part of the receptor. In both cases, the aim is to block growth-stimulating signalling, and thereby inhibit tumor cell proliferation (Rich J N, Bigner D D: Nat Rev Drug Discov 3: 430-446, 2004). There is, however, room for improvement. For example Iressa has proven to be a disappointment, acting in only a fraction of patients over-expressing the EGFR. For cetuximab, it still remains to be seen what will be the best chemotherapy combination treatment modality to increase the therapeutic impact of the treatment. These therapies can be combined with a radionuclide-based approach to kill tumor cells (Carlsson J, et al: Radiotherapy and Oncology, 66(2), 107-117, 2003), and one interesting example is the recent application of Gefitinib to modify the uptake and therapy effects of radio-labeled (astatinated) EGF (Sundberg A L et al: Eur J Nucl Med Mol Imaging 30: 1348-1356, 2003). Development of polypeptide anti-EGFR targeting agents provides an interesting alternative to the naturally agonistic (tumor-stimulating) biological EGF ligand, for the delivery of radionuclides for both diagnostic (imaging) and therapy purposes, as previously exemplified for HER-2 (Wikman M et al. Protein Engineering, Design & Selection (PEDS), 17(5), 455-462, 2004; Steffen A C et al. Cancer Biotherapy and Radiopharmaceuticals, 20, 239-248, 2005; Steffen A C et al. Eur J Nuclear Medicine, In press, 2005). Such polypeptides can also have biological effects, even without radioactivity, that are of interest for therapy. Z variants, also called “Affibody® molecules”, as disclosed for example in WO2005/0003156, are polypeptides which are intermediate in molecular weight (6-15 kDa), and can therefore have better penetration in tumor tissue than antibodies (150 kDa), and at the same time have better systemic circulation properties than low molecular weight substances like Iressa and Tarceva (≈1 kDa) which are rapidly eliminated via kidney excretion. In fact, Z variants typically have half-lives in a range suitable for in vivo imaging applications, and if needed for therapeutic or other applications, half-lives can be extended dramatically by gene fusion technology (see for example WO 2005/097202A).
Over-expression of EGFR is common in Head and Neck Squamous Cell Carcinomas, (HNSCC) (Rikimaru, K et al. Head Neck, 1992. 14(1): p. 8-13; Santini, J et al, Head Neck, 1991. 13(2): p. 132-9. Ekberg T et al. Int J Oncology, 26(5), 1177-1185, 2005). Increased levels of HER2 have been suggested in several studies of HNSCC (Craven, J. M et al. Anticancer Res, 1992. 12(6B): p. 2273-6), with possible prognostic value in oral Squamous Cell Carcinomas, (SCC) (Werkmeister, et al. Oral Oncol, 2000. 36(1): p. 100-5; Werkmeister, R. Am J Surg, 1996. 172(6): p. 681-3; Xia, W et al. Clin Cancer Res, 1997. 3(1): p. 3-9; Xia, W et al. Clin Cancer Res, 1999. 5(12): p. 4164-74). HER3 has been shown to be over expressed in HNSCC cell lines and associated with clinical malignant progression (Xia, W et al. Clin Cancer Res, 1999. 5(12): p. 4164-74; Shintani, S et al. Cancer Lett, 1995. 95(1-2): p. 79-83) and to be over expressed also in other types of malignancies (Gullick, W. J. Cancer Surv, 1996. 27: p. 339-49). Some human mammary carcinoma cell lines have HER4 transcripts (Plowman, G. D et al. Proc Natl Acad Sci USA, 1993. 90(5): p. 1746-50) but the role of HER4 in cancer is less clear (Srinivasan, R. et al. Cancer Res, 2000. 60(6): p. 1483-7). It is interesting to study co-expression of the four receptors, since it has been suggested that co-expression patterns may be associated with malignant phenotypes (Xia, W et al. Clin Cancer Res, 1999. 5(12): p. 4164-74; Bei, R. et al. J Pathol, 2001. 195(3): p. 343-8; Krahn, G. et al. Eur J Cancer, 2001. 37(2): p. 251-9). Immunohistochemical stainings of EGFR and HER2 have shown pronounced membranous staining. In contrast, HER3 and HER4 staining has been mainly cytoplasmic (Plowman, G. D et al. Proc Natl Acad Sci U S A, 1993. 90(5): p. 1746-50; Srinivasan, R. et al. Cancer Res, 2000. 60(6): p. 1483-7). Furthermore, EGFR and HER2 have been reported to express at high levels in both tumors and metastases. Thus, it seems that EGFR and HER2 are potential targets for macromolecular and peptide-based in vivo imaging and therapy applications while this might not be the case with HER3 and HER4.
Increased levels of EGFR-protein have also been found in urinary bladder carcinoma and the over-expression has been related to tumor stage and malignancy grade (Harney, J. V. et al, J Urol, 146, 227-31. (1991); Messing, E. M. Cancer Res, 50, 2530-7. (1990); Neal, D. E. et al, Cancer, 65, 1619-25. (1990); Sauter, G. et al. Int J Cancer, 57, 508-14. (1994); Gardmark T, et al. British Journal of Urology (BJU), 95, 982-986, 2005).
In Glioblastoma Multiforme (GBM) the most malignant form of the gliomas, which are common primary central nervous system tumors, over-expression of EGFR is detected in at least half of all analyzed tumors (Boskovitz A, et al. Expert Opin Biol Ther 4: 1453-1471, 2004; Shinojima N, et al. Cancer Res 63: 6962-6970, 2003; Ekstrand A J, et al. Cancer Res 51: 2164-2172, 1991; Rainov N G et al. Journal of Neuro-Oncology 35 13-28 (1997); Carlsson J et al. J. Neurooncol. 2005 Sep. 8; [Epub ahead of print]). The over-expression is due to gene amplification and/or increased transcription rates, and the number of 106 receptors per tumor cell has been reported (Rich J N, Bigner D D: Nat Rev Drug Discov 3: 430-446, 2004; Bigner S H et al. J Neuropathol Exp Neurol 47, 191-205 (1998); Carpenter G. Ann Rev Biochem 56, 881-914 (1987); Collins V P. Cancer Biology 4, 27-32 (1993); Libermann T A et al. Nature 313, 144-147, (1985); Kleihues P, Ohgaki H. Neuro-oncol 1, 44-51, (1999); Kleihues P, Ohgaki H. Toxicol Pathol 28, 164-170, (2000); Boskovitz A et al. Expert Opin Biol Ther 4, 1453-1471, (2004)). EGFR over-expression correlates with increased glioma growth rate and decreased survival (Rich J N, Bigner D D: Nat Rev Drug Discov 3, 430-446, (2004); Carlsson J et al. J. Neurooncol. 2005 Sep. 8; [Epub ahead of print]; Schlegel J et al. Int J Cancer 56, 72-77, (1994); Wikstrand C J, Bigner D D. J Natl Cancer Inst 90, 799-801, (1998); Shinojima N et al. Cancer Res 63, 6962-6970, (2003)) and it has been indicated that EGFR over-expression is most pronounced at the tumor cell invading edges (Okada Y, et al. Cancer Res 63, 413-416)(2003)). EFGR-specific binding polypeptides could potentially be employed for therapy applications for glioma therapy, for example, by locoregional administration into the postoperal cavity.
Several other malignancies of epithelial origin, such as those found in lung and breast, are also associated with a high expression of EGFR (Salomon, D. S. et al. Crit. Rev. Oncol. Hematol., 19(3):183-232, (1995)). EGFR receptors are also distributed among various normal tissues and expressed to rather high levels especially in liver hepatocytes and skin epithelium (Gusterson, B. et al. Cell Biol Int Rep, 8, 649-58. (1984); Damjanov, I. et al. Lab Invest, 55, 588-92. (1986)). This can potentially cause problems in therapy applications, especially radiotherapy, but is probably of less importance in diagnostic and imaging applications where low amounts of diagnostic or imaging markers which bind to EGFR receptors are given. Nevertheless, EGFR-binding polypeptides might find applications in certain cancers where local administration is to be considered.
It is an object of the invention to provide new EGFR-binding agents, that could be used for diagnostic, in vitro or in vivo imaging, as well as therapeutic applications. In addition, such EGFR binding polypeptides might find use in staging and as a direct assessment of SME based therapy aimed to down-regulate the target receptor.
In addition to the development of marketed molecular imaging agents, applications include use in the drug development and screening procedure where specific imaging agents are desired to measure outcome of treatment in in vivo models and subsequently during clinical development. Molecular Imaging provides a direct read-out of efficacy of a pharmaceutical aimed to down-regulate a growth factor receptor, as well as for assessing the anti-tumor effect.